Compositions and methods for obtaining nucleic acids from sputum

ABSTRACT

The present invention relates to compositions and methods for preserving and extracting nucleic acids from saliva. The compositions include a chelating agent, a denaturing agent, buffers to maintain the pH of the composition within ranges desirable for DNA and/or RNA. The compositions may also include a reducing agent and/or antimicrobial agent. The invention extends to methods of using the compositions of the invention to preserve and isolate nucleic acids from saliva as well as to containers for the compositions of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit from U.S. application Ser. No.60/386,397, filed Jun. 7, 2002, U.S. application Ser. No. 60/386,398,filed Jun. 7, 2002, and U.S. application Ser. No. 60/386,399, filed Jun.7, 2002, each of which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to compositions and methods forpreserving nucleic acids at room temperature for extended periods oftime and for simplifying the isolation of nucleic acids.

[0003] DNA can be extracted from virtually every type of cell in thehuman body, with the exception of red blood cells. The usual source ofbodily samples for extraction of DNA is venous blood, since the numberof nucleated white blood cells (principally neutrophils and lymphocytes)is relatively high and quite consistent: the normal range is about 5 to10 million white blood cells per milliliter of blood. The DNA content ofhuman cells is about 6 micrograms per million cells, so 1 milliliter cantheoretically yield from 30 to 60 micrograms of DNA. However, there areabout 5 billion red blood cells per milliliter of blood, which, sincethey contain no DNA, must be removed to obtain pure DNA. Furthermore,the use of blood as a source of DNA has many other disadvantages.Collection of blood is not a trivial procedure. Taking of venous bloodrequires trained personnel. It is an invasive procedure, whichfrequently causes some distress and pain to the donor. Precautions areneeded to minimize exposure of personnel to blood-borne pathogens. Oncecollected, the blood sample must be either frozen or quickly transportedto a laboratory for extraction of DNA. For these reasons, venous bloodis not the ideal source of DNA. A simpler procedure for obtaining bloodis to collect a few drops after a finger prick and blotting it onto apiece of filter paper. Less training of personnel is required. Oncedried, the DNA is quite stable. The amount of DNA recovered is small butsufficient for many forensic purposes. However, a finger prick is stillan invasive procedure and heme derived from hemoglobin in blood caninhibit some types of DNA analysis.

[0004] Swabbing the inside of the cheek with a brush (a buccal swab) isanother source of cells that contain DNA. It is much less invasive thantaking of blood and can be collected by individuals with less trainingthan is required in the collection of blood. Once collected, the timethat useable DNA can be recovered can be extended by either drying theswab or wiping onto filter paper and drying it. However, as the insideof the mouth is not a sterile source (as compared to blood) and microbescan degrade the quality of the DNA after a period of time. The number ofcells recovered by this procedure is not large and typically less than1-2 micrograms of DNA can be expected in the entire sample.

[0005] Saliva is a fairly clear, colorless fluid secreted principally bythe major salivary glands (parotid, submandibular, and sublingual). Itsfunction is to lubricate and cleanse the oral cavity, as well as toinitiate the process of digestion. The parotid gland primarily secretesserous (watery) saliva, while the other glands secrete a mixture ofserous and mucinous (sticky) saliva. Components of saliva includealbumin, globulin, mucins, and digestive enzymes. It has long been knownthat cellular DNA is present in saliva and that this DNA is suitable forforensic purposes. Forensic use is typically limited to victim orsuspect identification, using the tiny amounts of DNA from saliva thatmay recovered at a crime scene or from the back of a postage stamp. Thenotion that saliva may be a reliable source of genomic DNA and a rivalto venous blood samples for this purpose has been investigated morerecently in a scientific publication (van Schie, et al., J. Immunol.Methods 208:91-101, 1997). The authors used freshly collected or frozensaliva samples and purified the DNA by a fairly complex extractionprocedure. Estimates of the quantity of DNA recovered were based uponlight absorption at 260 nm, a procedure known to be an unreliable methodsince other common biological macromolecules, such as RNA, haveessentially the same ultraviolet light absorption spectrum.Nevertheless, these authors showed that quality genomic DNA was indeedpresent by gel electrophoretic analysis and polymerase chain reactionanalysis for certain allelic polymorphisms. Another communication(Terasaki, et al., Hum. Inmunol. 59:597-598, 1998) reported similarresults about the suitability of saliva as a source of DNA for HLAtyping by polymerase chain reaction analysis. Although the amount of DNArecovered was reported, the method used to measure DNA was not. Theseauthors provided 3 examples where saliva dried on filter paper yieldedDNA suitable for analysis.

[0006] With the increasing use of DNA-based analysis in forensics, lawenforcement, military, human medicine, veterinary medicine, andresearch, there is a need for a product that would allow saliva tobecome a standard reliable source of DNA from an individual (to replaceblood, the current standard). In forensic, military and mass disastersituations, for example, DNA samples are now routinely taken from livingpersons thought to be relatives of unidentified victims of accident orfoul play, to aid in identification of the dead. Military personnel orother individuals who expect to encounter hazardous situations wheretheir lives may be at risk may wish to store DNA samples prior toexposing themselves to these hazards. In the law enforcement area,convicted felons in both Canada and the United States are now requiredto provide DNA samples. DNA-based tests are expected to increase inmedicine, such as testing for cystic fibrosis, cytochrome P450 isotypes,polymorphisms affecting susceptibility to infectious and autoimmunediseases, HLA typing, paternity issues, to name but a few. In clinicalstudies, an example would be to screen populations for coloncancer-predisposing genes or family members of a breast cancer victimfor breast cancer predisposing genes. In all of these cases, there aresignificant advantages to providing a saliva sample rather thanproviding a blood sample as a source of DNA. All donors would preferdonating saliva rather than blood because of the discomfort, pain, orapprehension associated with phlebotomy or pin-pricks. Saliva has afurther advantage of not requiring specialized personnel therebyreducing cost where mass sample collection is being carried out. Therisk of blood-borne infection is likewise decreased.

[0007] In addition to the problem of developing a standard collectionand preservation method for DNA in saliva, there remains an ongoing needto improve methods of overcoming problems specific to the recovery ofnucleic acids from saliva. The problem of extraction of high molecularweight DNA and RNA from mammalian cells has been partially addressed byBirnboim in Methods of Enzymology 216:154-160, 1993, but this work wasnot extended to the recovery of nucleic acids from mucin-containingbodily fluids.

[0008] Multimeric proteins called mucins are high molecular weightglycosylated proteins that form a major part of a protective biofilm onthe surface of epithelial cells, where they can provide a barrier toparticulate matter and bind microorganisms. These glycoproteinscontribute greatly to the viscoelastic nature of saliva. The majorhigh-molecular-weight mucin in salivary secretions is MUC5B, one of fourgel-forming mucins that exist as multimeric proteins with molecularweights greater than 20-40 million daltons. MUC5B is a large oligomericmucin composed of disulphide-linked subunits.

[0009] It is known that reagents that reduce disulfides also reduce theviscosity of mucin, such as that found in sputum or saliva. Reducingagents, in particular sulfur-containing chemicals such asβ-mercaptoethanol and dithiothreitol, are widely used in biochemistry.However, many biochemically relevant reducing agents are capable ofreacting in solution with dissolved oxygen. This is known areautooxidation (also called autoxidation or auto-oxidation), where1-electron reduction intermediates of oxygen are formed, viz.,superoxide (O₂ ⁻.), hydrogen peroxide (H₂O₂) and hydroxyl radical (OH.).In addition, transitional metal cations function as catalysts and O₂ ⁻.has been demonstrated to be an intermediate. Unfortunately, reducingagents and reducing compositions of the prior art have a relativelyshort shelf life, especially in basic solutions, and stock solutionsthat contain reducing agents cannot be prepared and stored under ambientconditions for an extended period time, usually not more than a day ortwo.

[0010] Therefore, in addition to a need for a means to collect sputum orsaliva, and subsequently preserving the nucleic acids contained thereinby contacting them with a stabilizing composition, there is a need forthe inclusion of a stable reducing agent into the composition, such thatnucleic acids can be conveniently recovered from it, especially afterextended periods of time in the presence of oxygen at neutral or mildlyalkaline pH.

SUMMARY OF THE INVENTION

[0011] The present inventor has developed a composition, which, whenmixed with a mucin-containing bodily fluid, preserves the nucleic acidsat room temperature under ambient conditions for extended periods oftime. There is no requirement for freezing of the samples before nucleicacid recovery and purification. The properties of this composition arethat it (a) chemically stabilizes nucleic acids, (b) inhibits nucleasesthat may be present in the saliva, and (c) is compatible withproteolytic enzymes and other reagents used to purify/amplify oligo- orpolynucleotides. A fourth and novel property of this composition is thatit contains an agent that rapidly reduces the viscous properties ofmucin, greatly facilitating the extraction of nucleic acids containedwithin.

[0012] Accordingly, a first aspect of the invention features acomposition for preserving nucleic acids that includes a chelatingagent, and a denaturing agent, where the pH of the composition isgreater than 5.0. In one embodiment, the composition is an aqueoussolution.

[0013] In another embodiment, the composition also includes a reducingagent. For example, it can include one or more of the following:ascorbic acid, dithionite, erythiorbate, N-acetylcysteine, cysteine,glutathione, dithiothreitol, 2-mercaptoethanol, dierythritol, aresin-supported thiol, a resin-supported phosphine, vitamin E, andtrolox, or salts thereof. Desirably, the reducing agent is ascorbicacid, erythiorbate, N-acetylcysteine, dithiothreitol, or2-mercaptoethanol, and most desirably, the reducing agent is ascorbicacid. In another embodiment, the composition does not contain ascorbicacid. In yet another embodiment, the concentration of the reducing agentin the composition is greater than or equal to 50 millimolar.

[0014] Antioxidant free-radical scavengers are also desirable reducingagents for the composition of the present invention. Examples includeantioxidant vitamins, antioxidant hormones, antioxidant enzymes, thiols,and phenols.

[0015] Desirably, the reducing agent retains reducing activity for atleast 46 days in the presence of one or more of the following: oxygen,ambient air, ambient light, and alkaline pH.

[0016] The chelating agent of the composition can be selected from thegroup consisting of: ethylenediamine tetraacetic acid (EDTA),cyclohexane diaminetetraacetate (CDTA), diethylenetriamine pentaaceticacid (DTPA), tetraazacyclododecanetetraacetic acid (DOTA),tetraazacyclotetradecanetetraacetic acid (TETA), and desferrioximine, orchelator analogs thereof. Desirably, the chelating agent is cyclohexanediaminetetraacetate (CDTA), diethylenetriamine pentaacetic acid (DTPA),tetraazacyclododecanetetraacetic acid (DOTA), or desferrioximine, andmost desirably, the chelating agent is cyclohexane diaminetetraacetate(CDTA).

[0017] In another embodiment, the chelating agent of the compositioninhibits metal redox cycling. By “inhibits metal redox cycling” is meantthe inhibition of metal-based oxidation/reduction cycles that producereactive oxygen free-radical species. Examples of redox ion pairsinvolved in such cycles include Fe²⁺/Fe³⁺, Cu¹⁺/Cu²⁺, and variousoxidation states of molybdenum, vanadium, nickel, and cobalt. Chelatorsthat bind one or both ions of a redox ion pair can inhibit theproduction of reactive oxygen species such as, for example, hydroxylradical (HO.), hydroperoxyl radical (HOO.), superoxide radical (O₂ ⁻.),nitric oxide radical (NO.), or peroxynitrite radical (ONO₂ ⁻.).

[0018] The nucleic acid to be preserved by the composition can be DNA orRNA, including mRNA or viral RNA.

[0019] The pH of the composition can between from about 5.0 and about11.0, desirably from about 6.5 to about 7.5, and most desirably, about7.0. For the preservation of DNA, a pH from about 7.0 to about 10.0 canbe used. Depending on other components of the compositions, desirablepHs are about 7.5, about 8.0, or a pH range from about 8.0 to about 9.0.A buffer, such as HEPES, TRIS, or carbonate buffer can be added to thecomposition to maintain the pH in a constant range. For the preservationof RNA, a pH from about 5.0 to about 7.0, desirably from about 6.5 toabout 6.8 can be used. Again, a buffer, such as BES, can be used tomaintain the pH in a constant range.

[0020] The denaturing agent of the composition can be selected from thegroup consisting of: urea, dodecyl sulfate, guanidinium chloride,guanidinium thiocyanate, perchlorate, and an alcohol. Desirably, thedenaturing agent is urea, dodecyl sulfate, or an alcohol, wherein thealcohol is 10%-60% of the total composition volume. The alcohols can bemethanol, ethanol, n-propanol, isopropanol, n-butanol, trifluoroethanol,phenol, or 2,6-di-tert-butyl-4-methylphenol.

[0021] In another embodiment, the composition includes an antimicrobialagent. By “antimicrobial agent” is meant a substance or group ofsubstances which reduces the rate of growth of an organism compared tothe rate of growth of the organism in their absence. A reduction in therate of growth of an organism may be by at least 5%, more desirably, byat least 10%, even more desirably, by at least 20%, 50%, or 75%, andmost desirably, by 90% or more. The definition also extends tosubstances which affect the viability, virulence, or pathogenicity of anorganism. An antimicrobial agent can be natural (e.g., derived frombacteria), synthetic, or recombinant. An antimicrobial agent can bebacteriostatic, bactericidal or both. An antimicrobial agent isbacteriostatic if it inhibits cell division without affecting theviability of the inhibited cell. An antimicrobial agent is bactericidalif it causes cell death. Cell death is commonly detected by the absenceof cell growth in liquid growth medium (e.g., absence of turbidity) oron a solid surface (e.g., absence of colony formation on agar). Those ofskill in the art know that a substance or group of substances which isbacteriostatic at a given concentration may be bactericidal at a higherconcentration. Certain bacteriostatic substances are not bactericidal atany concentration. Desirably, the composition of the invention includesan alcohol as an antimicrobial agent, and most desirably the compositionincludes ethanol.

[0022] In another embodiment, the composition also includes an inhibitorof ribonuclease. Desirable inhibitors are selected from the groupconsisting of: heparin, heparan sulfate, oligo(vinylsulfonic acid),poly(vinylsulfonic acid), oligo(vinylphosphonic acid), andpoly(vinylsulfonic acid), or salts thereof. The inclusion of aninhibitor of ribonuclease in the composition of the invention isparticularly desirable when the nucleic acid to be preserved is RNA,desirably mRNA, or when the nucleic acid to be preserved is from a virusor a bacterium.

[0023] A second aspect of the invention features a method of reducingthe viscosity of a mucin-containing bodily fluid or tissue by reducingdisulfide bonds inherent to mucin, wherein the bodily fluid or tissue ismixed with a composition of the invention that includes a reducingagent. In one embodiment, the bodily fluid is sputum, desirably saliva.By “sputum” is meant that mucoid matter contained in or discharged fromthe nasal or buccal cavity of an animal, including saliva and dischargesfrom the respiratory passages, including the lungs. In anotherembodiment, the method includes the recovery of a nucleic acid.

[0024] A third aspect of the invention features a method of preserving anucleic acid contained in sputum that includes the steps of obtainingsputum from a subject, and contacting the sputum with a composition ofthe invention, thus preserving the nucleic acid.

[0025] In one embodiment, when the nucleic acid is DNA, the DNA isstable for more than 14 days, desirably more than 30 days, and moredesirably more than 60 days. In another embodiment, when the nucleicacid is DNA and the composition does not contain ascorbic acid, the DNAis stable for more than 60 days, and desirably more than 360 days.

[0026] A fourth aspect of the invention features a method of recoveringa nucleic acid from sputum that includes the steps of: i) obtainingsputum from a subject, ii) contacting the sputum with a composition ofthe invention to form a mixture, iii) contacting the mixture with aprotease, and iv) recovering the nucleic acid from the mixture.Desirably, the protease is proteinase K or pronase.

[0027] In one embodiment of any of the second, third, or fourth aspects,the sputum is saliva. In another embodiment, the sputum is from amammal, desirably a human. In yet another embodiment, the nucleic acidis DNA or RNA. If the nucleic acid is RNA, desirably it is mRNA or viralRNA. The nucleic acid can be from a source foreign to the subject fromwhich the sputum sample is taken. For example, the nucleic acid can befrom a bacterium or a virus that is residing in the buccal, nasal, orrespiratory passages of the subject.

[0028] In a fifth aspect, the invention features a method of preservingand/or recovering a nucleic acid from a bodily fluid that includes,placing the bodily fluid into a first region of a container, placing acomposition of the invention into a second region of the container,which is separated from the first region by a barrier, closing thecontainer, and disturbing the integrity of the barrier such that thecomposition and the bodily fluid are brought into contact.

[0029] In one embodiment, the disestablishment of the barrier is coupledto the closing of the container when a lid is placed on it. In oneexample, the barrier is punctured. In a desirable example, the barrieris in the form of a pivoting sealing disc. In this example, attachmentof the lid to the container forces the disc to pivot from its originalposition of spanning the space between the first region and the secondregion to a position in which both regions are exposed to each other,thereby forming a mixture between a composition of the invention and thebodily fluid is allowed. Desirably, the bodily fluid is sputum, and mostdesirably, saliva.

[0030] In a sixth aspect, the invention features a device for preservingand/or isolating a nucleic acid obtained from a biological sample. Thedevice includes: a container that has a first region for collecting abiological sample and a second region containing a composition forpreserving a nucleic acid, a barrier between the first region the secondregion that keeps the biological sample and the composition separate, ameans for closing the container, and a means for disturbing theintegrity of the barrier such that the composition is capable ofcontacting the biological sample. The first region can have an openingof from 2.0 to 7.0 cm, desirably from 2.5 to 3.5 cm, and most desirably3.0 cm. Desirably, the biological sample is sputum, and most desirably,saliva.

[0031] In one embodiment of the sixth aspect, the nucleicacid-preserving composition is a composition of the present invention.In another embodiment, the means for closing the container is coupled tothe means for disturbing the integrity of the barrier. In yet anotherembodiment, the means for closing the container is an airtight lid.

[0032] In a seventh aspect, the invention features a method ofmanufacturing a device for preserving a nucleic acid in a biologicalsample that includes: providing a container that has a first region anda second region, with the first region suitable for containing acomposition of the invention and the second region having an openingsuitable for the application of a biological sample; placing thecomposition into the first region; and applying a barrier to thecontainer between the first region and the second region, with thebarrier being impermeable to the composition and capable of beingdisestablished.

[0033] In an embodiment of either the sixth or seventh aspect, thebarrier can be a pivoting disc, where in a first position, the discspans the compartment and separates the first and second areas. Pivotingthe disc to a second position (e.g., by connecting a screw-on lid to aplunger mechanism which contacts the disc, causing it to pivot when thelid is screwed on) disestablishes the barrier and allows the biologicalsample contained in the first region to contact the composition that iscontained in the second region.

[0034] By “about” is meant ±10% of the stated value or a chemical orobvious equivalent thereof.

[0035] By “alcohol” is meant a water-miscible organic compoundcontaining a hydroxyl group, including water-miscible mixtures ofhydroxyl-containing organic compounds.

[0036] By “antioxidant free-radical scavenger” is meant a substance thatreduces a reactive oxygen free radical species. Such free radicalsinclude, for example, hydroxyl radical (HO.), hydroperoxyl radical(HOO.), superoxide radical (O₂ ⁻.), nitric oxide radical (NO.), orperoxynitrite radical (ONO₂ ⁻.).

[0037] By “aqueous solution” is meant a solution or suspension thatcontains 30% or more water by volume.

[0038] By “bodily fluid” is meant a naturally occurring fluid from ananimal, such as saliva, serum, plasma, blood, urine, mucus, gastricjuices, pancreatic juices, semen, products of lactation or menstration,tears, or lymph.

[0039] By “biological sample” is meant any sample containing nucleicacids that has been obtained from or deposited by an animal.Non-limiting examples include skin, hair, bodily fluids, fecal matter,and tissue.

[0040] By “chelator analog” is meant a derivative chelator compound withthe same backbone structure and having the same general properties asthe parent chelator compound.

[0041] By “denaturing agent” is meant a substance that alters thenatural state of that to which it is added.

[0042] By “mucin” is meant any mucoprotein that raises the viscosity ofthe medium surrounding the cells that secrete it.

[0043] By “mucoid” is meant any bodily fluid containing mucin By“nucleic acid” is meant a chain of the nucleotides, includingdeoxyribonucleic acid (DNA) or ribonucleic acid (RNA), typically foundin chromosomes, mitochodria, ribosomes, bacteria, or viruses.

[0044] By “nucleic acid-preserving composition” is meant any compositionof the present invention, unless otherwise specified.

[0045] When referring to a nucleic acid, by “stable” is meant that atleast about 50% of the initial amount of high molecular weight nucleicacid (DNA, RNA, mRNA, or viral RNA) contained in a sample is stillpresent after storing the sample at ambient temperature (i.e., 20° C. to25° C.) for the specified time period. The amount of high molecularweight DNA in a sample can quantified by densitometry analysis of thehigh molecular weight DNA band from an agarose gel (see FIG. 1 andExample 4).

[0046] By “resin-supported phosphine” is meant a polymer that contains amultiplicity of covalently-bound phosphine groups.

[0047] By “resin-supported thiol” is meant is meant a polymer thatcontains a multiplicity of covalently-bound sulfhydryl groups.

[0048] By “saliva” is meant the secretion, or combination of secretions,from any of the salivary glands, including the parotid, submaxillary,and sublingual glands, optionally mixed with the secretion from thebuccal glands.

[0049] By “sputum” is meant that mucoid matter contained in ordischarged from the nasal or buccal cavity of a mammal, including salivaand discharges from the respiratory passages, including the lungs.

[0050] By “subject” is meant any animal. Desirably, the subject is amammal that can produce saliva for the purposes of nucleic acidextraction. Most desirably, the subject is a human.

[0051] Other features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0052]FIG. 1 is an electrophoresis agarose analysis of DNA isolated fromsaliva using the capacity of methods of one embodiment of the invention.

[0053]FIG. 2 is a graph illustrating real time PCR of stimulated salivaDNA of Example 5.

[0054]FIG. 3 is a graph illustrating real time PCR of unstimulatedsaliva DNA of Example 6.

[0055]FIG. 4 is an electrophoresis agarose analysis of the DNA in salivasamples mixed with compositions of the invention, the mixtures havingbeen incubated for various times at various temperatures.

[0056]FIG. 5 shows structures of (oxidized) ascorbate anion, (reduced)dehydroascorbic acid, and a free radical intermediate FIG. 6 is acompilation of two spectrophotometric scans of sodium ascorbate (100 μM)in CB (1 mM CDTA, 10 mM BES, pH 7.4), prepared under aerobic conditionsover 30 minutes at room temperature (scan 1) and 3 minutes afteraddition of a few crystals of MnCl₂.(scan 2), as per Example 8.

[0057]FIG. 7 is a compilation of spectrophotometric scans, at theindicated times, of the 100 μM sodium ascorbate prepared in CB ofExample 8. The solution was exposed to ambient atmosphere andtemperature between scans but was not contacted with MnCl₂ (see Example9).

[0058]FIG. 8 is a graph of absorbances at 265 nm, obtained at theindicated times, of a solution of sodium ascorbate (250 mM) containing30 mM Tris-HCl, pH 8.0, 30% ethanol, 3 mM CDTA, mixed with 50 mL of CB,as per Example 10. The stock solution was maintained at room temperatureand no precaution was taken to exclude ambient atmosphere or ambientlight.

[0059]FIG. 9 is a compilation of spectrophotometric scans of the 46day-old solution prepared in Example 10. Scan 1 (t=46 days) was takenbefore the addition of MnCl₂. Scan 2 was taken 2 minutes after theaddition MnCl₂. Scan 3 was taken 8 minutes after the addition MnCl₂.Scan 4 was taken 27 minutes after the addition MnCl₂.

[0060]FIG. 10 is an exploded view of a sample container of theinvention. Included in the figure is a cross-sectional top view taken atline 1-1 of container 3 showing plunger 4 and plunger channel 5. Alsoshown is a cross-sectional top view taken at line 2-2 of container 3,showing supports 6 for sealing disc 7 (not shown in this figure butshown in FIG. 11).

[0061]FIG. 11 is a side view of the sample container of FIG. 10, nowshowing sealing disc 7.

DETAILED DESCRIPTION

[0062] The following standard abbreviations are used herein: DNA,deoxyribonucleic acid; RNA, ribonucleic acid; mRNA, messenger RNA;HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; BES,N,N-bis[2-hydroxyethyl]-2-aminoethane-sulfonic acid; TRIS,tris(hydroxymethyl)aminomethane, CDTA, cyclohexane diaminetetraacetate;DTPA, N,N-bis(2-(bis(carboxymethyl)amino)ethyl)glycine; DOTA,1,4,7,10-tetrazacyclododecanetetraacetic acid; and TETA, 1,4,8,11-tetraazacyclotetradecanetetraacetic acid.

[0063] Compositions of the Invention

[0064] The present inventors have developed compositions that rendersputum as a viable option to the use of blood as a source of nucleicacids. The compositions provide the advantageous properties of chemicalstabilization of nucleic acids and the inhibition of nucleases,including deoxyribonucleases, and microbial growth. Chemicalstabilization of the nucleic acids in a saliva sample is achievedthrough the use of buffers to maintain an appropriate pH, as well as theuse of chelating agents to prevent the phenomenon of metal redox cyclingor the binding of metal ions to the phosphate backbone of nucleic acids.The chelating agents of the invention also participate in the inhibitionof deoxyribonucleases and microbial growth, which can be additionallyinhibited by the inclusion of denaturing agents and/or other suitableantimicrobial substances, such as ethanol, into the compositions of theinvention. The compositions of the invention can also include one ormore reducing agents, which can reduce sample viscosity, thereby makingnucleic acid recovery an easier process.

[0065] Accordingly, the present invention features a composition forpreserving and/or recovering nucleic acids from sputum, desirablysaliva, that includes one or more chelators and one or more denaturingagents, wherein the pH of the composition is greater than 5, desirablywithin a pH range of about 6 to about 11, more desirably within a pHrange of about 7.5 to about 10.0, and most desirably, within a pH ofabout 7.0.

[0066] The chemical backbone and the purine bases of DNA are most stableat slightly alkaline pH, with an optimal stability generally recognizedas being within a pH range of about 7-11, and desirably a pH of about 8.Below a pH of about 6, depurination (i.e., spontaneous loss of purinebases from the deoxyribose-phosphate backbone) can occur. Above a pH ofabout 10, spontaneous loss of amino groups from cytosine nucleotides mayoccur, thereby converting cytosine to uracil. Above a pH of about 12,DNA is denatured, converting it from the double-strand form to thesingle-strand form. In contrast, RNA is most stable in the pH range of5.0 to 7.0, desirably a pH of from 6.5 to 6.8. Accordingly, the pH ofthe composition may be adjusted using pH buffers, desirably those thatbest control the pH within the range of about 5 to about 11. Examples ofpH buffers with desirable properties include, but not limited to, TRIShydrochloride, HEPES and BES.

[0067] DNA has a strong affinity for metal ions, some of which, such asthe common transition metals iron or copper, can catalyze the formationof reactive oxygen species. Therefore, a composition of the inventionincludes one or more chelators that can form complexes with metal ionsto prevent them from binding to DNA, remove metal ions that that havealready bound to DNA, or bind to metal ions (e.g., Fe(II)/Fe(III) orCu(I)/Cu(II)) strongly enough to inhibit their redox cycling, and hence,the formation of reactive oxygen species. EDTA, a commonly used chelatorin biological reagents, can be of some use for either of these purposes.More desirable are stronger chelators (i.e., chelators with a higherdissociation constant than EDTA when bound to a metal), used alone or incombination, that include, but are not limited to, CDTA, DTPA, DOTA,TETA, and desferioximine, or chelator analogs thereof. The amount orconcentration of chelator will depend upon the strength of the chelator,which would need to be determined empirically. For CDTA, concentrationsin the 1-20 mM range are sufficient, however other concentrations wouldwork, and the compositions of the invention are not intending to belimited to this range.

[0068] Deoxyribonucleases and ribonucleases are enzymes that breakdownDNA or RNA, respectively. Their main source in the digestive tract issecretions of the pancreas, although lower levels may be present incells of the salivary gland and buccal mucosa. In addition,microorganisms resident in the mouth or from recently ingested foods maycontain deoxyribonucleases or ribonucleases. Pancreaticdeoxyribonuclease is known to require divalent metal ions such asMg(II), Mn(II) and/or Ca(II) for enzymatic activity. The strongchelators described above, in addition to providing chemical stabilityto the nucleic acids, will inhibit this class of metal ion-requiringdeoxyribonucleases. The action of deoxyribonucleases and ribonucleasescan also be inhibited by denaturing agents that will destroy the complexstructures of these enzymes (proteins). Hence, denaturing agents areincluded in the nucleic acid preserving composition of the invention.Examples of denaturing agents that may be used (alone or in combination)include, but not limited to, urea, soluble salts of dodecyl sulfate andother strong detergents, guanidinium chloride, guanidinium thiocyanate,soluble salts of perchlorate, alcohols, such as ethanol, above 10%.Other reagents, such as heparin, heparan sulfate, or oligo(vinylsulfonicacid) (Smith, et al., J. Biol. Chem. Mar. 20, 2003; [epub ahead ofprint]) are known to inhibit the action of deoxynucleases and/orribonucleases.

[0069] Low specificity proteases such as proteinase K are frequentlyused in the purification of nucleic acids. Since proteases arethemselves proteins, their action can be inhibited by denaturing agents.Thus, a balance must be struck between the concentration of denaturingagents that will, on the one hand, inhibit deoxyribonucleases orribonucleases and denature other proteins in saliva and, on the otherhand, not significantly inhibit the proteolytic enzymes. At later stagesin DNA purification, the DNA is often concentrated by precipitation withalcohol. Thus, salts, buffers, chelators and other components of thenucleic acid preserving/recovery solution must be chosen so as not toprecipitate when concentrations of alcohol over 50% are added toprecipitate the DNA.

[0070] The viscosity of sputum and saliva depends upon the presence ofvery high molecular weight glycoproteins complexes called mucins,particular the gel-forming mucins (Offner, et al., Adv. Dent. Res.14:69-75, 2000; Seregni, et al., Tumori 83:625-632, 1997). It has beenfound that the inclusion of a reducing agent into a composition of theinvention has the effect of markedly reducing the viscosity of saliva,especially “unstimulated” saliva, thereby facilitating the recovery ofnucleic acids. Accordingly, in one embodiment, a composition of theinvention further includes one or more reducing agents. The reducingagents are desirably at high concentration (greater than 0.05 M). Whilenot wishing to be limited by theory, it is presumed that the reducingagent reduces the viscosity of the saliva by breaking disulfide bondsthat hold together chains of mucin, and that any reducing agent that hasthe appropriate redox potential to reduce disulfide bonds in proteinswould be suitable. Desirably, the reducing agent is selected from thegroup consisting of: ascorbic acid, dithionite, erythiorbate,N-acetylcysteine, cysteine, glutathione, dithiothreitol,2-mercaptoethanol, dierythritol, a resin-supported thiol, aresin-supported phosphine, vitamin E, and trolox, or salts thereof.

[0071] In another embodiment, a composition of the invention thatincludes a reducing agent maintains reducing capacity at roomtemperature in a sealed container in the presence of ambient oxygen,and/or in the presence of ambient light for more than a week, desirablyfor up to about 46 days, and most desirably for at least 46 days. Thisembodiment combines the nucleic acid stabilization provided by a strongchelator a denaturing agent, and a reducing agent in a composition witha pH within the range of about 6 to about 11, and desirably a pH ofabout 8.0.

[0072] A particularly desirable reducing agent is sodium ascorbate. Aswell as an important dietary antioxidant micronutrient, ascorbic acid(vitamin C) is a non-thiol reducing agent and is inexpensive, non-toxic,and stable in the presence of the chelators and denaturing agents thatare included in the compositions of the invention. The structures of(oxidized) ascorbate anion, (reduced) dehydroascorbic acid, and a freeradical intermediate are shown in FIG. 5. The most thoroughly studiedoxidation reaction of ascorbate is its oxidation by oxygen. As with manyother reducing agents, trace amounts of transitional metals such as ironor copper can promote autooxidation (Buettner, Free Radic. Res. Commun.1:349-53, 1986; Buettner and Jurkiewicz Radiat.Res. 145:532-41, 1996;Miller, et al., Free Radic. Biol. Med. 8:95-108, 1990). Metalcation-catalyzed oxidation of ascorbate can be conveniently monitored asa decrease in absorbance at 265 nm (Buettner Free Radic. Res. Commun.10:5-9, 1990), as described in Example 8 and shown in FIGS. 5, 6, and 8.Certain chelating agents can appreciably slow down autooxidation ofascorbate at pH 7.0 or lower (Buettner J. Biochem. Biophys. Methods16:27-40, 1988), as described in Example 10 and shown in FIG. 8.

[0073] In another embodiment, a composition of the present inventionincludes one or more chelators, one or more denaturing agents, and oneor more antimicrobial agents, wherein the pH of the composition iswithin a pH range of about 6.0 to about 11.0, desirably at a pH of about8.0. Microbial growth may also be inhibited by the strong chelators anddenaturing agents, for example, ethanol, described above. Therefore, ina further embodiment of the present invention, a composition forpreserving and/or recovering DNA from sputum includes one or morechelators and one or more denaturing agents, wherein at least one ormore of the denaturing agents and/or chelating agents is present inamounts to act as an antimicrobial agent.

[0074] Reagents that indicate when a biological sample has beencontacted with a composition of the invention can also be included aspart of the composition. Desirable are those reagents that result in avisual color change of the composition solution upon mixing with theadded sample. These reagents can function by reacting with any number offunctional groups that are contained in biological samples, including,for example, amines, thiols, or glycosyl groups. Such colorimetricreagents are known to those skilled in the art and are chosen in such amanner that other components of the composition do not interfere withtheir effective usage.

[0075] Methods of the Invention

[0076] The present invention features methods of collecting, preserving,and recovering nucleic acids from sputum using a composition of theinvention. The methods of the invention involve contacting a sputumsample from a subject with a composition of the invention and optionallymixing the resulting solution with a protease, such as pronase orproteinase K. Furthermore, some compositions of the invention feature areducing agent that can facilitate the recovery of nucleic acids fromcomposition/sample mixtures by decreasing the viscosity of thesemixtures.

[0077] Accordingly, one aspect of the invention features a method ofpreserving a nucleic acid contained in sputum that includes the steps ofobtaining sputum from a subject, and contacting the sputum with acomposition of the invention, thus preserving the nucleic acid. Examples1 and 2 describe the collection of saliva, both from subjects that canfollow instructions and from those that can not.

[0078] The sputum is typically contacted with a composition of theinvention upon collection or immediately after it is collected, andpreferably not much later than about 1 hour after collection. This timecan vary depending on storage conditions of the sputum after collection.For example, it could be indefinite if stored frozen or perhaps 1-2 daysif stored at 4° C. A reducing agent can be in the preserving compositionused, or added at a later time prior to nucleic acid isolation.Desirable reducing agent-containing compositions are those that arestable and retain a reducing capacity for more than a week, desirablyfor up to about 46 days, and most desirably for at least 46 days.

[0079] In an example (see Example 5), the results of which are presentedin Table 1, saliva was collected and mixed with approximately an equalvolume of a composition of the invention (see Example 3 forpreparation), and analyzed for DNA content by PCR analysis at latertimepoints. TABLE 1 Estimated amounts of DNA in saliva samples* Donor #1 2 3 4 5 6  7 8 9 10 11 Stim. saliva collected on 02Feb26, analyzed 64days by the DNase method 21.2 21.4 16.6 16.0 28.8 44.8 22.2 16.6 Unstim.saliva collected on 02Mar25, analyzed 15 days later by DNase method 64.280.6 24.4 27.2 69.0

[0080] To collect the sputum from the subject it is preferred that themouth be rinsed before sampling. Food particles can introduce foreignDNA and saliva transferred by kissing can be a source of foreign humanDNA. The mouth can be rinsed with about 50 mL of tepid water by vigorousswishing or by brushing with a tooth brush without tooth paste.Unstimulated saliva is usually of the mucinous type and is secreted at aslow rate. Stimulated saliva (anticipation of tasty food, sweet or sourcandy) is of the serous (watery) type and secreted at a faster rate. Ithas been found (see Table 2) that there is more DNA in 2 mL ofunstimulated saliva than 2 mL of stimulated saliva. After rinsing of themouth and waiting about two or three minutes, the donor may spit avolume (for example, about 2 mL) of “unstimulated” saliva into thereceiving tube. If this proves to be difficult, saliva flow canconveniently be stimulated with a cube of table sugar, or any other suchsaliva-stimulatory substance that does not interfere with DNA recoveryor purification. TABLE 2 Comparison of DNA content of unstimulated andstimulated saliva Donor #7 unstimulated stimulated Collected on 2002April 6, analyzed 36.2* 21.8* 2 days later by the DNase method

[0081] Another aspect of the invention features a method of reducing theviscosity of a mucin-containing bodily fluid or tissue by reducingdisulfide bonds inherent to mucin, wherein the bodily fluid or tissue ismixed with a composition of the invention that includes a reducingagent. In one embodiment, the bodily fluid is sputum, desirably saliva.

[0082] Yet another aspect of the invention features a method ofrecovering a nucleic acid from sputum that includes the steps of: i)obtaining sputum from a subject, ii) contacting the sputum with acomposition of the invention to form a mixture, iii) contacting themixture with a protease, and iv) recovering the nucleic acid from themixture.

[0083] Suitable proteases include, for example, proteinase K or pronase.The protease may suitably be in a dry form that would become activatedonce mixed with sputum and a composition of the invention. In oneembodiment, the protease is deposited onto an interior surface of thecollection device. This can be accomplished by dissolving the proteasein a solution made up of equal volumes of 5% sucrose in water and 5%glycerol in ethanol and then, after placing the solution on the surface,removing the volatiles under a controlled vacuum to leave the proteasebound to the surface as a sticky residue. If the composition does notcontain a reducing agent (or even if it does), a reducing agent can beadded at any time prior to isolation of the nucleic from the sample,desirably prior to or concurrently with contacting the sample with asuitable protease.

[0084] When sputum is mixed with a composition of the present invention,cells are disrupted, nucleic acids are liberated from the cells,membranous material is solubilized, proteins are stripped from thenucleic acids, and protein digestion begins. If present, a reducingagent in the composition reduces the viscosity of the gel-forming mucin.Incubation can be at room temperature over a relatively long period oftime (days or weeks) while samples are being shipped to a laboratory foranalysis. If transferred to a laboratory soon after collection,incubation at 55° C. for 4 to 16 hours is sufficient to allow theactivated protease to digest the majority of protein to small peptidesor amino acids. Under such conditions, nucleic acids and polysaccharidesremain relatively intact.

[0085] Once digestion is complete, nucleic acid isolation can beperformed using any technique known in the art (Short Protocols inMolecular Biology, 5th Edition Frederick M. Ausubel, Roger Brent, RobertE. Kingston, David D. Moore, J. G. Seidman, John A. Smith (Editor),Kevin Struhl (Editors). ISBN: 0-471-25092-9. 2002. John Wiley and Sons).In one example, in which SDS is used as a denaturant component of thecomposition, a “precipitation solution”consisting of, for example,potassium chloride may be added to a portion of the sputum-compositionmixture resulting in the precipitation of potassium dodecyl sulfate,after standing on ice to cool the solution. Following a short period ofcentrifugation to remove the precipitate and any residual insolublematerial, the supernatant is collected. At this stage, the supernatantis expected to contain as much as 10 -30 nanograms per microliter ofDNA. For analyses where as little as 1 nanogram of DNA is sufficient,the sample can be diluted.

[0086] When larger amounts of DNA are required, the DNA in thesupernatant can be precipitated by the addition of alcohol andredissolved in any suitable buffer. This step has the effect of removinginhibitory components of the composition, which are present to preservethe nucleic acids during transport to the laboratory.

[0087] If more highly purified DNA is required, then other knownpurification steps can be used (Short Protocols in Molecular Biology,5th Edition Frederick M. Ausubel, Roger Brent, Robert E. Kingston, DavidD. Moore, J. G. Seidman, John A. Smith (Editor), Kevin Struhl (Editors).ISBN: 0-471-25092-9. 2002. John Wiley and Sons), such as extraction withphenol or solid-phase extraction. It should be noted that, because theDNA is in a relatively pure state using the procedures described above,any additional purification steps are made easier when compared toanalogous purifications of DNA originating from a blood sample.

[0088] The methods of the present invention can be used to isolatenucleic acids from sputum for any application requiring a nucleic acidsample. For example, some specific applications of the methods of thepresent invention include, but are not limited to, forensicapplications, medical applications (including genetic screening anddisease typing), and paternity testing.

[0089] Another aspect of the invention features a method of preservingand/or recovering a nucleic acid from a bodily fluid that includes,placing the bodily fluid into a first region of a container, placing acomposition of the invention into a second region of the container,which is separated from the first region by a barrier, closing thecontainer, and disturbing the integrity of the barrier such that thecomposition and the bodily fluid are brought into contact. Collectiondevices of the invention, which also can serve as containers for bringthe compositions and nucleic acid-containing bodily fluids together aredescribed below.

[0090] Collection Devices

[0091] The invention also provides a novel collection device useful forcollecting a biological sample from a subject, and subsequently mixingthe collected sample with a composition intended to stabilize, preserve,or facilitate the recovery of components of the sample. Such componentsmay include, without limiting the invention, nucleic acids, proteins,peptides, toxins, chitins, fatty acids, and glycogens. Non-limitingexamples of biological samples are skin, hair, fecal matter, bodilyfluids, and tissue.

[0092] Desirably, the invention features a device for preserving and/orrecovering a nucleic acid obtained from a biological sample. The deviceincludes: a container that has a first region for collecting abiological sample and a second region containing a composition forpreserving a nucleic acid, a barrier between a first region and a secondregion that keeps the sample and composition separate, a means forclosing the container, and a means for disturbing the integrity of thebarrier, such that the composition is capable of contacting the bodilysample. In one embodiment, the composition is a composition of thepresent invention. In another embodiment, the sample is a biologicalfluid.

[0093] The collection device of the invention simultaneously servesseveral functions. Some of the desirable features of this collectionvessel include one or more of the following:

[0094] a) it may be constructed of a sturdy breakage-resistant plastic,desirably a biocompatible plastic. Desirably, the container would beconstructed from a material that would not leach chemicals into thecontainer's contents;

[0095] b) it would have a broad mouth that would make it relativelysimple for a subject to place the required volume of fluid sample,desirably expectorated sputum, and most desirably expectorated saliva,into the device's container;

[0096] c) the bottom part of the container would be narrow to reduce theoverall volume of the container to make it easier to collect the smallvolume (1-2 milliliters) of fluid that would be expected from a routinesampling, in particular, when the sample is an expectorate. Optionally,the device would contain markings to allow for an estimate of the samplevolume collected;

[0097] d) the means for closing the container may be a cap that isdesigned to lock once tightened to become tamper-resistant;

[0098] e) the means for closing the container may be a cap that isdesigned to provide a liquid-tight and/or airtight seal for thecontainer once the cap is fixed into place;

[0099] f) the barrier may be a septum or plastic bag compartment thatwould separate the composition from the fluid until the septum or bagcompartment is pierced or the contents otherwise released;

[0100] g) the barrier may be in the form of a pivoting partition. Inthis embodiment, attachment of the lid to the container forces thepartition to pivot from its original position of spanning the spacebetween the first region and the second region to a position in whichboth regions are exposed to each other and contact between thecomposition contained in one space and the bodily fluid contained in theother space is allowed;

[0101] h) the barrier can be press fit, glued, or heat fit into place;

[0102] i) the means for closing the container may be coupled to thedisestablishment of the barrier; and

[0103] j) an antimicrobial agent that coats the outside of the device.

[0104] A device of the invention is shown in FIGS. 10 and 11. With cap 1not attached to the device, a biological sample (not shown) is appliedto a first region 8 of container 3, which is separated from a secondregion 9 by sealing disc 7. After sample application, cap 1 is placedonto the device and secured via a screw thread mechanism to a tight fit,thereby sealing container 3. As the cap is twisted on (shown by dottedline and arrow 10, ram 2, which is attached to cap 1, moves downward asshown by dotted line arrow 11. This downward movement forces plunger 4,which is contained in plunger barrel 5, downward as indicated by dottedline and arrow 12. The downward movement of plunger 4 forces sealingdisc 7 to pivot, as shown by dotted line and arrow 13. Pivoting of disc7 disestablishes the barrier between regions 8 and 9, thereby permittingcontact between the sample and a composition of the invention, shown asa dotted solution contained in region 9.

[0105] Kits

[0106] The present invention also features kits for performing themethods of the invention that include a device of the inventioncontaining a composition of the invention, with instructions forstabilizing, preserving, or facilitating the recovery of nucleic acidsfrom a biological sample by using the device to bring a biologicalsample into contact with the composition.

EXAMPLES Example 1

[0107] Protocol for Obtaining Saliva Samples from Subjects Capable ofFollowing Instructions.

[0108] The subject is instructed to wait for a period of 20-30 minutesbefore last eating. The subject will brush his teeth without usingtoothpaste, if possible. The subject will rinse his mouth vigorouslywith 50 mL of cool or tepid water. The subject will then spit salivainto the special collection tube until the level of saliva reaches the 2mL mark. This may take several minutes. If the subject finds that he isunable to deliver sufficient saliva, he will be given a cube of tablesugar to chew, and told not to be concerned if some of the sugar is spitinto the tube.

[0109] When the required amount of saliva is collected, it is mixed with2 mL of a nucleic acid-preserving composition. The precise way this willbe introduced will depend upon the container design.

[0110] Once the composition is introduced, the cap is attached to thecontainer and tightened to seal it securely. The container is thenvigorously shaken and the process is complete. The DNA is now in anintermediate preserved state. It can be maintained in a frozen state orat any temperature up to about 60° C.

[0111] The container can be mailed back to the testing lab at roomtemperature.

Example 2

[0112] Protocol for Obtaining Saliva Samples from Babies, Very YoungChildren and Infirm Adults Incapable of Following Instructions.

[0113] A rubber or plastic tube or nipple will be introduced into themouth, attached to a sponge, suction bulb or small syringe, and kept inthe mouth for several minutes until visible drooling occurs. A bit ofsugar cube will be placed in the mouth to stimulate saliva if necessary.The responsible adult will wear disposable gloves provided for thepurpose to avoid contamination with his/her DNA. The responsible adultwill draw saliva into the bulb or syringe and transfer it into thecollection container. The DNA preserving/extraction composition isintroduced and the container is capped and sealed. The tube isvigorously shaken for 1 minute.

Example 3

[0114] Preparation of a Nucleic Acid-Preserving Composition.

[0115] The composition of the nucleic acid-preserving solution used inExamples 4-6 is 33 mM TRIS-HCl, 0.67 M urea, 0.67 M LiCl, 0.6% sodiumdodecyl sulfate, 3.3 mM CDTA, 30% ethanol, and 0.25 M sodium ascorbate,all adjusted to a final pH of 8.0. In the examples, the composition ismixed with an equal volume of saliva. Subsequent to these experiments,it has been found that a composition which is 0.3 M TRIS-HCl, 0.67 Murea, 0.67 M NaOAc, 0.6% sodium dodecyl sulfate, 3.3 mM CDTA, 30%ethanol, and 0.1 M sodium ascorbate, all adjusted to a final pH of 8.0,stabilizes DNA for longer periods of time.

Example 4

[0116] Extration of Minimally Purified Chromosomal DNA from theStimulated Saliva of 8 Different Donors.

[0117] After collection of saliva in an equal volume of the compositionas noted in Example 3, followed by 14 days storage at room temperature,a 0.25 mL portion of each donor's sample was treated with proteinase K,centrifuged briefly to remove insoluble material and the DNA therein wasprecipitated with 2 volumes of ethanol. The precipitate was dissolved in0.05 mL of water, and an 8 μL aliquot (equivalent to about 20 μL ofundiluted saliva) was analyzed by electrophoresis on a 0.8% agarose gel,stained with ethidium bromide to visualize the DNA (see FIG. 1). Of noteis the characteristic band of chromosomal DNA present in all samples atthe position of the arrow, that corresponds to the position ofchromosomal DNA extracted from white blood cells (data not shown).

Example 5

[0118] “Real Time” Polymerase Chain Reaction Using DNA from StimulatedSaliva.

[0119] Stimulated saliva samples collected on Feb. 26, 2002 (seeTable 1) and stored at room temperature were analyzed 62 days later.Minimally purified DNA was prepared as follows: an aliquot wascentrifuged to remove insoluble material; to the clarified supernatantwas added 2 volumes of ethanol; the precipitate containing DNA wascollected by centrifugation and redissolved in water. A volume of theredissolved DNA equivalent to 0.05 microliters of each of the originalsaliva samples was used for analysis. Real time PCR was carried outusing a Roche Light Cycler instrument, where the fluorescent dye SYBRgreen I was added to follow the reaction (see results of FIG. 2). Theprimers were designed to detect the human Clotting Factor IX gene(Grant, et al., J. Immunol. Methods 225:61-6, 1999). C=control, highlypurifed white blood cell DNA. Each curve represents results using salivaDNA from different donors, represented by a number. These results usingreal time PCR demonstrate the suitability of minimally purified salivaDNA from different donors for PCR analysis.

Example 6

[0120] “Real Time” Polymerase Chain Reaction Using DNA from UnstimulatedSaliva.

[0121]FIG. 3 is a graph showing saliva DNA samples collected on Mar. 25,2002 (see Table 1) and analyzed on 30 days later in accordance withFIG. 1. Minimally purified DNA was used Polymerase chain reaction andother conditions as described in Examples 4 and 5 except salivacollection was done under unstimulated conditions. Numbers refer toindividual donors. C is control DNA, a highly purified sample of DNApurified from blood.

[0122] Tables 1 and 2 show estimates of DNA recovered from salivasamples. In all cases, the individual donor has been identified by aunique number. These data show that the amount of DNA that can berecovered from this group of donors ranges from 16 micrograms permilliliter of saliva and higher. Estimation of the amount of DNA bychemical methods such as DABA presents some problems and the DNasemethod provides most reliable results.

Example 7

[0123] Stability Studies on DNA from Saliva.

[0124] Saliva was mixed with an equal volume of the indicatedcomposition and the mixture was incubated for the indicated time periodat the indicated temperature (see Table 3). After incubation,approximately 40 μL of mixture was digested briefly with ribonuclease toremove the majority of the RNA present in the sample, then applied tothe indicated lane of a 0.8% agarose gel. Following electrophoresis, thegel was stained with ethidium bromide as in Example 4. TABLE 3 Lane No.Composition Incubation Conditions 1 0.5 M NaOAc, 0.2 M TRIS-HCl, 0.15 M70° C. for 3 days, then Na ascorbate, 10 mM CDTA, 1% SDS, 50° C. for 16days 30% (vlv) ethanol, pH = 9.5 2 0.5 M NaOAc, 0.2 M TRIS-HCl, 10 mM50° C. for 21 days CDTA, 1% SDS, 30% (v/v) ethanol, pH = 9.5 3 0.5 MNaOAc, 0.2 M TRIS-HCl, 10 mM 70° C. for 3 days, then CDTA, 1% SDS, 30%(v/v) ethanol, pH = 50° C. for 31 days 9.5 4 0.67 M LiCl, 33 mMTRIS-HCl, 0.67 M 20° C.-25° C. for 15 urea, 0.6% SDS, 3.3 mM CDTA, 30%months (v/v) ethanol, pH = 8.0 5 0.67 M LiCl, 33 mM TRIS-HCl, 0.67 M 20°C.-25° C. for 15 urea, 0.6% SDS, 3.3 mM CDTA, 30% months (v/v) ethanol,pH = 8.0 6 Control chromosomal DNA prepared from white blood cells

Example 8

[0125] Rapid Autooxidation of Ascorbate in the Presence of a TransitionMetal Ion.

[0126] A solution of sodium ascorbate (100 μM) in CB (10 mM BES, pH 7.4,containing 1 mM CDTA) was freshly prepared under aerobic (equilibratedwith ambient air) conditions. Several spectrophotometric scans over 30minutes at room temperature showed no change in the absorbance profile(all similar to scan (1)). Scan (2) was taken 3 minutes after additionof a few crystals of MnCl₂. The results can be seen in FIG. 6. As shown,100 μM ascorbate at neutral pH has an absorbance (λ_(max)=265 nm) ofabout 1.25 (corresponding to the expected molar extinction coefficient(AM) of about 12,500. Upon addition, the transition metal, manganouschloride, catalyzed the autooxidation of ascorbate, which canconveniently be monitored by a decrease in absorbance at λ=265 nm(Buettner, Free Radic. Res. Commun. 10:5-9, 1990).

Example 9

[0127] Spontaneous Autooxidation of Ascorbate.

[0128] Repeated scans at the indicated time points were taken of analiquot of the 100 μM sodium ascorbate solution prepared in Example 8,before the addition of MnCl₂. The sample was exposed to air andmaintained at room temperature between scans. The results areillustrated in FIG. 7, and indicate that autooxidation of ascorbateoccurs at pH 7.4 can occur over an extended period of time in thepresence of low concentrations (1 mM) of CDTA, a “strong” chelator.

Example 10

[0129] Stability of Sodium Ascorbate in a Nucleic Acid-PreservingComposition.

[0130] A stock solution of sodium ascorbate (250 mM) was prepared in asolution containing 30 mM Tris-HCl, pH 8.0, 30% ethanol, 3 mM CDTA. 20μL was removed at the indicated times, mixed with 50 mL of CB (seeExample 8) and the absorbance at 265 nm was read immediately. The stocksolution was maintained at room temperature. The results are shown inFIG. 8.

[0131] While the present invention has been described with reference towhat are presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

[0132] All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

What is claimed is:
 1. A method of preserving a nucleic acid containedin sputum comprising the steps of: a. obtaining sputum from a subject;and b. contacting said sputum with a composition comprising a chelatingagent and a denaturing agent, wherein the pH of said composition isgreater than 5.0, thus preserving said nucleic acid.
 2. The method ofclaim 1, wherein said sputum is saliva.
 3. The method of claim 1,wherein said sputum is from a mammal.
 4. The method of claim 1, whereinsaid nucleic acid is DNA or RNA.
 5. The method of claim 4, wherein saidnucleic acid is DNA.
 6. The method of claim 5, wherein said pH isbetween 7.0 and 10.0, inclusive.
 7. The method of claim 4, wherein saidnucleic acid is mRNA or viral RNA.
 8. The method of claim 7, whereinsaid pH is between 5.0 and 7.0, inclusive.
 9. The method of claim 5,wherein said nucleic acid is stable for more than 14 days.
 10. Themethod of claim 9, wherein said nucleic acid is stable for more than 60days.
 11. The method of claim 10, with the proviso that said compositiondoes not contain ascorbic acid.
 12. The method of claim 11, wherein saidnucleic acid is stable for more than 360 days.
 13. A method ofrecovering a nucleic acid from sputum comprising the steps of: a.obtaining sputum from a subject; b. contacting said sputum with acomposition comprising a chelating agent and a denaturing agent, whereinthe pH of said composition is greater than 5.0, to form a mixture; c.contacting said mixture with a protease; and d. recovering said nucleicacid from said mixture.
 14. The method of claim 13, wherein said sputumis saliva.
 15. The method of claim 13, wherein said nucleic acid is DNAor RNA.
 16. The method of claim 15, wherein said RNA is mRNA or viralRNA.
 17. A method of preserving a nucleic acid from a bodily fluidcomprising: i. placing said bodily fluid into a first region of acontainer; ii. placing a composition comprising a chelating agent and adenaturing agent, wherein the pH of said composition is greater than5.0, into a second region of said container, said second regionseparated from said first region by a barrier; iii. closing saidcontainer; and iv. disestablishing said barrier such that saidcomposition contacts said bodily fluid forming a mixture, therebypreserving said nucleic acid.
 18. The method of claim 17, furthercomprising recovery of said nucleic acid from said mixture.
 19. Themethod of claim 17, wherein said bodily fluid is sputum.
 20. The methodof claim 17, wherein said bodily fluid is saliva.